Effects of Six Different Agars on Tracheary Element Differentiation in Explants of Lactuca

全文

(1)Efects Of Six Diferent Agars on Tracheary Element]Diferentiation in Explants of」 Lα ε′ πεα Sango Baba* and Lorin W. Roberts**. ABSTRACT The relat市 e effect市 eness of s破 different agars(1%w/V)fOr ser宙 ng as a supporting matrix during the auxin― cytokinin induction of xylogenesis in Lα θ ι πθ α explants was exarnined. The conllnercial agar preparations included E)ifco Bacto―. Agar, Gibco Phytagzr, KC― TC agar, Flow agar, FWC Sea Plaque, and FMC SeaPrepo No signiflcant differences in numbers of tracheary elements differen_ tiated were detected in explants incubated on media solidined with Bacto― Agar,. Phytagar,and Flow agar, respectively. One experiment involving KC― TC agar. produced unusuaHy high dumbers Of tracheary elements.. Tracheary element. counts were signincantly less in an explants cultured on media containing either SeaPlaque or SeaPrep agarose.. INTRODUCTION Agar gel has been traditional supportive agent for serrlisolid plant. tissue culture media.. Agar is not physiologicaHy inert,. and this. natural product contains varying amounts of growth― stilnulating and growth― inhibiting contanlinants (Huang and Murashige, 1976; Bonga. 1982; Dodds and Roberts.1982).The grOss chemical analysis of Difco. agars has been published (Pierik, 1971).. Few investigators have. examined the possible role of agar as a nutritive source in plant tissue. culture media. *. Romberger and Tabor(1971)repOrted that an. Konan Women's Conege, Morikitacho 6-chome, Higashinada―. “agar. ku, Kobe City. 658 Japan **. Departinent of Blological Sciences, University of ldaho, Moscow, Idaho 83843, U.So A..

(2) 4露. Effects of Six Different Agars on To E.Direrentiation in Lα. θ ι αθ α. inhibitory effect'' was considerably decreased by autoclaving the agar. mediurrl with sucrosee. Activated charcoal has been used to adsorb. organic and inorganic molecules from culture media, and this agent. may remove inhibitory contarninants from the agar gel(Kohlenbach and Wernicke, 1978). Because of the variety of plant tissue culture agars that are now. commerciany availablee a series of experiments were conducted in order to test the relative effectiveness of several agars in acting as a supportive matrix for explants exhibiting xylogenesise. Since this was a. vere made to isolate any physio‐ preliminary investigationo no efforts 、 logicaHy―. active contarninants from the agars.. Because of numerous. studiese the induction of xylogenesis in lettuce pith explants was chosen as a test systeⅡ l(Dalessandro and Roberts。 1971; Roberts, 1976).. MATERIALS AND METHODS Explants were prepared frorn the core of pith excised from heads of cornrnerciany― grown. グ υα L.Romana)as Romaine lettuce (Lα θ′πθα sα ι. described by Dodds and Roberts(1982). After surface sterilization in e rinses in 10% (V/V)C10rox for 10 min followed by three success市 vater, borings of the pith parenchyma were with sterile doubledistilled 、 a 5Π lln. I.D. cork borero. The pith cylinders were then cut into exロ. plants approxirnately 21nrrl in thicknesso. The explants were rinsed. twice with sterile double― distilled water, blotted on sterile Whatlnan No. 1 ■lter paper,and transferred individuaHy to glass vials(151■. containing 10 nll of a xylem― induction mediumo. l capacity). The medium consisted. of a Murashige and Skoog (1962) basal Salt nlixture, suppplemented with πノθ―inositol(100 mg/1), thiamineO HCI(0。 l mg/1),glyCine(2。. 0. mg/1),nicotinic acid(0。 5 mg/1),pyridoxine・ HCI(0。 5 mg/1),IAA(10 mg/1),kinetin(0。 l mg/1),D― glucose(2%チ w/v),and One of the agars (1%チ. W/V)liSted belowo The agal・ s employed in the present study. included Difco Bacto― Agar,Gibco Phytagar, KC― TC agar,Flow agar, 1). FMC Seaplaque, and FMC SeaPrep. The pH of the rnedium was adiuSted t0 5。. 7,and the complete medium was sterilized by autoclave..

(3) 479. Sango Baba and Lorin W. Roberts. The vials,capped with KimKap c10sures,were dark incubated at 25°. C. for 7 dayso Each agar was tested at least three times with a minimum of 10 samples for each treatinent.. Data were analyzed using Duncan's. NIultiple Range Test on the Statistical Analysis SysteHl at the University of ldaho。. Tracheary element cen counts were performed on the explants after 7 days of culture by a modiflcation of the Brown and Rickless (1949). maceration technique (See Dodds and Roberts。. 1982).. Explants were. placed individually in vials containing l rnl of a maceration. ■uid. con_. sisting of equal parts of chromium trioxide(5%w/v)and HCl(5% V/V) fOr 24 hr at room temperatureo. The maceration luid was then. removed and replaced with l nll of distilled water.. The sample was. drawn repeatedly into a syringe (2 ml) equipped with a 22-guage needleo The sample was brought to a total volume of 2 rnl, thoroughly Πlixed. by syringe, and l. ■1l. of the sample. 、 vas. transferred to a. Sedgwick― Rafter plankton counting chambero The number of tracheary. elements was counted in each of 7 optical ields with the aid of a Whipple eyepiece lnicrometer at 100x magnincation. The total number of tracheary elements in each explant was calculated (see Dodds and Roberts.1982).. RESULTS AND DISCUSSION No signincant differences in tracheary element count per explant was found between Difco Bacto― Agar, Gibco Phytagar, and Flow agar. (Table l).The KC一 TC agar produced a considerably greater number of tracheary elements in one of the three experirnents in which it was testedo. This anomalous result could be due to the lack of an inhibitory. substance, the presence of some xylogenic promoter, or tO SOme 1)Difco Laboratories,P.Oo Box 1058A,Detroit,Ⅳ I1 48232,Uo S.A。 Gibco Laboratories,3175 Staley Road,Grand lsland,NY 14072,Uo S.A. KC Biological lnc。 ,Po. Oo Box 14848,Lenexa,KAN 66215,U.S.A.. Flow Laboratories lnc.,7655 01d Springhouse Road,McLean,VA 22102,Uo S.A. FMC Corporation,lⅦ arine Colloids Division, BioProducts Departlnent, 5 Maple Street,Rockland,ME 04841,Uo S.A..

(4) 侶θ. Effects of Six Different Agars on T.Eo Di∬. ισ α erentiation in Lα θ. “ Table lo The effects of s破 different agars on the in duction of tracheary l). πθ α element cytodifferentiation in explants of Lα σι. Tracheary Elements(x103) Experiment Number. Agar preparation(1%W/V). Difco Bacto― Agar. Gibco Phytagar Flow agar. 10。. 5a. 10。. 6a. 13。 3a. 15。. KC― TC agar. 9b. 13.2a. Fゝ江C. SeaPlaquc agarose Fゝ江C SeaPrep agarose. 19.3a 17.4ab. 8。. 9b. O。. 5c. 13。 2a 6a. 13。. 3a. 12.4a. 23。 3c. 14.Oa. 16。. 9.4d. 6.2b. 5e. O.01c. l。. 1) Each agar was tested at least three times with a minimum of 10. explants for each treatinent.. Thus, tracheary element counts. represent a mean of 10 samples. Treatlnents not fonowed by the same letter are signincantly dif_ ferent at the 5ッ多level, as deterrnined by]Duncan's Multiple Range Test.. unknown variable in techniqueo. The inconsistency in the. data. suggests that additional experiments should be performed with ]KC― agare. TC. Signi■ cantly smaHer numbers of tracheary elements were dif‐. ferentiated in the presence of the two agaroses in comparison to the other. agars tested. 15°. SeaPrep has a low gening temperature (apprOximately. C), and the medium containing this agarose had a viscous liquid. consistency during incubation of the explants.. This property may have. been inhibitory to the initiation of xylogenesiso. SeaPlaque, on the. other hand, gels completely in less than 10 min at temperatures below 25°. C. Since both Fn/1C)agaroses were highly inhibitory to xylogenesis,. some additional factor inluencing cyto―. differentiation must be involved。. Similar inhibitory effects on xylogenesis in lettuce pith explants were. found using an agarose (Type A) obtained frorn Calbiochem― Behring. prior to the present study (unpublished, Roberts).. Agarose is a. purined linear galactan hydrocoHoid isolated from agar, and it was devised for the diffusion and electrokinetic movement of biopolymers. (Guiseley and Renn, 1977). 2)CalbiOCherrl― Behring,P.0。. A hypothesis can be advanced that thё. Box 12087,San Diego,CA 92112.

(5) Sango Baba and Lorin Wo Roberts. 盤】. typical tissue culture agar does play a nutritive role in plant tissue culture media, and that this role(s) iS largely elirninated during the. removal of anionic polysaccharides and/Or Other constituents in the preparation of agaroseo A review of the purincation procedures em‐ ployed in the manufacture of agarose is available frorn Fヽ江C Corporation. (Guiseley and Renn,1977)。 The authors express thanks to E)ro Donald W. Renn,Senior iResearch Fellow,FttЛ C. Corporation, for the samples of SeaPlaque and SeaPrep. and for his interest in this proiecto. We also appreciate the assistance. of KC Biological,Inc.for the sample of KC― TC agare References ｀ Huang,Lo C。 ,and T。 M[urashige.1977。. Plant tissue culture media:maior. constituents, their preparation and some applications.. θCπ πrθ И[ssθ. Tissπ. Jι. _. グづ α′ θη几イαππαJ 3: 539-548。 θ Bonga,Jo M. 1982. Tissue culture techniques. In Tづ. ssπ. πFarθsι ,7, θCπ πrθ づ Jι. ed.Jo M.Bonga and DoJ.Durzan,pp.4-35。 lMartinus NijhoL The Hague. jπ θ ′Tお sπ θCπ π″ πι Sづ πPJα π γ Dodds`J.H。 ,and Lo Wo Roberts。 1982.Eχ 夕θ Jι. .. Carnbridge University Press. Pierik,R.L.M.1971. Plant tissue culture as lnotivation for the symposium。 jθ γ ι グ θ η′Mc′ jα ,ed. J. van Braft, s 9/Sι θ づ πCθ ttpο πθ πι sづ π Ⅳπ γ In E//aθ ι Jグ Zα ι η θ. D.A..A.Ⅳ [ossel,Ro Lo M.Pierik,and H.Veldstra,pp.3-13。. H,Veenman&. Zonen N.V。 ,Wagenlngen. s shoot apical meri_ ααbグ θ Romberger,J.A.,and Co A.Tabo■ 1971.The P′ θθ. stem in culture.Io Agar and autoclaving effects.ス Kohlenbach, Ho W。 , and Wo Wernicke.. 1978。. π.工. Bθ ′ 。58:131-140.. Investigations on inhibitory. effect of agar and function of active carbon in anther culture. Z. P//α. πZθ π‐. pλノ sグ θ J. 86: 463-472.. DalessandrO, G。 ,and Lo Wo Roberts.. 1971.. Induction of xylogenesis in pith. parenchyma explants of Lα θ ι π .ス πo J.Bθ ム 58:378-385。 “ Robers,Lo W.1976.Cガ θごSSt“ πι J〔シS′ ι π。グ Sづ sα ]屁 )α θ αι づ η P″ πι se x夕 Jθ多7π θ θηづ Carrlbridge University Press.. Murashige,T。 ,and F. Skoog。 1962. A revised mediurrl for rapid growth and bioassays with tobacco tissue cultures. l101. Brown,R。 ,and P.A.Rickless.1949。. Pんノsづ οJ.PJα πι 。15:473-497.. A new method for the study of cell.

(6) 482. Effects of Six Different Agars on T.E. Differentiation in Lα θ′ zθ α division and cell extension with prelirninary observations on the effect of o Rθ ノ。Sθ a B 13δ :110-125. ternperature and nutrients.P“ θ. 1977.ス gα Юsθ. jθ ι ガθ π,P%の θ γ s,α π α “ Biomedical Applications. FMC Corporation,Rockland,A/1E.. (lo Guiseley,K.B。 ,and Do Wo Renn。. f Paγ が.

(7)